miR-1 1scr 206 206scr

RD

GAPDH

PAX3

37kDa

53kDa

Figure S1: Western blot of RD (ERMS) cell shows that ectopic

miR-206 (20nM) decreased PAX3 expression in RD cell but not

in Rh18 cells, compared with scrambled control (206scr). No

significant change was observed in miR-1 transfected cells.

37kDa

53kDa

Rh18

GAPDH

PAX3

miR-1 1scr 206 206scr

Supplementary Information – 5

figure S2: A) qRT-PCR on Rh30 cells shows that transfection with miR-1/-206

precursors (pre) but not scrambled controls (scr) result into increased expression

of miR-1/-206. B) qRT-PCR on JR1 and Rh30 cells validate successful

transfection of miR-29 precursors.

A

B

Sham 29a 29b 29c mimic ctrl

RD

34kDa

37kDa

CCND2

GAPDH

Rh18

34kDa

37kDa

CCND2

GAPDH

Sham 29a 29b 29c mimic ctrl

Figure S3: Western blot shows that ectopic miR-29a /b/c

(20nM) decreased CCND2 expression in RD (ERMS) cells,

compared with sham control or negative miRNA precursor

control. While in Rh18 (ARMS) cells, transfection of miR-29c but

not miR-29a/b down regulated CCND2 expression compared

with controls.

Rh18

GAPDH

E2F7

37kDa

97kDa

Sham miR-29a miR-29b miR-29c mimic ctrl

Sham miR-29a miR-29b miR-29c mimic ctrl

RD

GAPDH

E2F7

37kDa

97kDa

Figure S4: Western blot of RD (ERMS) cell and Rh18 (ARMS)

cell. Ectopic miR-29c / -29a (20nM) decreased E2F7 expression

in Rh18 cells, compared with sham control or negative miRNA

precursor control. No significant change was found in RD cells.

A

B

Rh30 withCell s (%)

live apoptoticcontrol 77.65 8.27miR29a 73.75 16.38miR29b 67.14 18.67miR29c 71.95 17.19

Rh30 withcells (%)

G1 S G2/MControl 45.15 36.29 17.10miR-29a 45.49 36.27 15.90miR-29b 44.54 43.45 9.57miR-29c 42.44 40.02 15.78

C

(i) (ii) (iii) (iv) (v)

(i) (ii) (iii) (iv) (v)

Figure S6a: Functional assays in Rh30 cells transfected with miR-29a, -29b, or -29c (20nM). Apoptosis assay (A) showed significant pro-apoptotic effect of miR-29a (ii), -29b (iii) or -29c (iv) in serum-starved Rh30 compared to negative miRNA control (i). Proliferation assay (B) showed no significant effect caused by individually transfected miR-29 family members. Cell cycle assay (C) showed no significant changein Rh30 cells transfected with miR-29a (ii), -29b (iii) or -29c (iv) compared to negative miRNA control (i). Data are shown in (v). Negative miRNA precursor control#1 (Ambion, 20nM) serves as control.

A

B

(i) (ii) (iii)

Rh30 withcells (%)

G1 S G2/Msham 41.88 36.07 18.46miR-29s 46.57 23.72 25.66

C

Figure S6b : Cell function assay of Rh30 cells transfected with combined miR-29a, -29b,

and -29c (10nM for each). A) Apoptosis assay shows pro-apoptotic effect in miR-29

transfected Rh30 cells (ii) compared to control cells (i), and data are presented in (iii). B)

Proliferation assay show no effects of combined miR-29 family members transfected Rh30

cells compared to control cells. C) Cell cycle assay shows mild G1 arrest (ii) compared to

control (i), and data are showed in (iii).

(i) (ii) (iii)

Rh30 withCell s (%)

live apoptoticSham 83.78 13.4miR-29 76.01 19.56

R-PE R-PE

1948 -2119

1948 CAGTGGTCCC AATAGGAGAC AAAGGAGAGT GATTGATTTT 1987 1988 CTTCCTCCAA TAGTTGGTTT CAAATCCTTT TGAACACGTT 2027 2028 CGACAAAAGC AGTGGAGAAG AGGAAGACCT GGAGCAATAA 20672068 AAGACAAATG CAACATTTTA AGGCAATGGT TTCACATGGT 21072108 TACATATCAA AA 2119

Figure S7a: Deletion in PAX3 3’UTR in JR1 (ERMS) cells. Sequencing electropherogram of the PAX3-3’UTR sequence points the deletion site and the deleted sequence in the 3’UTR is shown in the inset.

PAX3 transcript

Sequence that does not showing here

Coding: 382-1833

3’UTR: 1834-3356

2158 and 3133: miR-1/206 binding sites

Missing regions

Mutation

Translocation

Figure S7b: Schematic figure showing the 5’ terminus of 3’UTR of PAX3 transcript in RMS

cells as well tissue samples. A)~E) According to the sequencing result, most of the missing

regions happen among the 1830-2127 of transcript variant I. This region, so called “hot

zone”, is around 31-156 nts upstream of the first miR-1/206 binding site (2158). Sequencing

result also show that F) point mutations. G) shorter transcript. H) Fusion PAX3-FOXO1 due

to translocation t(2,13)

PAX3-3’UTR WT + + + - - - - - -

PAX3 3’UTR Del1830-2002 - - - + + + - - -

PAX3 3’UTR Del1948-2119 - - - - - - + + +

miR-1 mimic - + - - + - - + -

miR-206 mimic - - + - - + - - +

* * * *

*

Figure S7c: Luciferase reporter assay show that the lucifrease expression from

wildtype PAX3 3’UTR sGG vector (WT) is repressed due to co-transfected miR-1/-

206, and the deletion mutant PAX3 3’UTR which missing the 1948-2119nt region

manages an escaping from the repression of miR-1/-206, while another deletion

mutant which missing the1830-2002nt region fails to do so.

ctrl 29a 29b 29c ctrl 29a 29b 29c

Figure S8: Transcript level of myogenesis genes including alpha actin,

myogenin, myoD1 and troponin T were checked with realtime PCR in RMS cells

before and 48h after miR-29 transfection. Among them, α actin was increased

after miR-29 transfection in Rh30 and RD cells, and myogenin was up-regulated

after ectopic expression of miR-29 in RD cells. We noticed that there was no

expression of myogenin in both ARMS cell lines with (Rh30) or without (Rh18)

PAX3-FOXO1 translocation. * p<0.05, vs negative miRNA precursor control.

* *

**

* *

* *

JR1 Rh30

ctrl 29a 29b 29c ctrl 29a 29b 29c ctrl 29a 29b 29c ctrl 29a 29b 29c

RD Rh18

ctrl 29a 29b 29c ctrl 29a 29b 29c

JR1 Rh30 RD Rh18